Cyclic AMP/PKA‐Mediated Regulation of Mitochondria and Branched‐Chain Amino Acid Metabolism in S49 Lymphoma Cells

Abstract

The role of mitochondria in responses mediated by the second messenger cAMP acting via protein kinase A (PKA) is poorly understood. To assess such a role, we used quantitative proteomic analysis of mitochondria‐enriched fractions and performed functional and morphological studies of wild‐type (WT) and kin‐ (PKA‐null) murine S49 T lymphoma cells. We found increased abundance of 110 proteins, including 8 involved in branched‐chain amino acid (BCAA) metabolism, in WT, but not kin‐, S49 cells incubated for 16 hr with the cell‐permeable cAMP analog 8‐CPT‐cAMP (CPT). CPT also increased mRNA expression of genes involved in BCAA metabolism in WT, but not kin‐, S49 cells. In addition, CPT enhanced survival of WT cells (but not kin‐ cells or WT cells in the presence of the PKA inhibitor H89) grown in media lacking glutamine. Studies using a Seahorse XF analyzer revealed that CPT enhanced the maximal rate of respiration and the capacity of WT cells to oxidize BCAA, while diminishing glycolytic flux (indicated by extracellular acidification). Basally, mitochondria of WT and kin‐ S49 cells have similar structural features but 16 hr incubation of WT (but not kin‐) cells with CPT produced changes of apoptosis (e.g. decreased mitochondrial density and size, and loss of cristae). Together, these findings show that cAMP, acting via PKA, regulates BCAA metabolism, mitochondrial function and structure. Regulation of mitochondria thus likely contributes to cAMP/PKA‐mediated cellular responses. Support: HHMI (S.S.T.), NIH DK54441 (S.S.T.) and GM066232 (P.A.I.)